Environmental factors influencing sporocarp formation in <Emphasis Type="Italic">Typhula ishikariensis</Emphasis>

Environmental factors influencing sporocarp formation in Typhula ishikariensis were studied under controlled conditions. Sporocarp formation in T. ishikariensis was divided into two stages: stipe elongation from the sclerotium and fertile head development at the tip of the stipe. Factors required for each stage differed. At the stipe elongation stage, low temperature (10°/5°C; day/night) and high humidity were important, but light was not required. In contrast, at the fertile head stage, light and moderate day length (8h/day) were essential. Fertile heads developed at 46µEm^–2s^–1; and high intensity (137µEm^–2s^–1) did not suppress development. Moreover, adding unsterilized soil to the sea sand medium accelerated sporocarp formation. These findings imply that the sclerotium of T. ishikariensis recognizes several physical factors for sporocarp formation. Sporocarps of T. ishikariensis developed within 4 weeks after incubation under optimal conditions. The sporocarp produced basidiospores, and differential mating incompatibility was confirmed among monokaryons derived from basidiospores produced under artificial conditions. This method should be useful for obtaining monokaryons for genetic studies of T. ishikariensis.     

Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells

Oligodeoxynucleotides containing the cytosine-phosphate-guanine (CpG) motif (CpG-ODNs) have been shown to induce T(H)1 immune responses in animals. Since the sequences of CpG-ODNs that induce T(H)1 responses are considered to vary among animal species, it is necessary to identify effective CpG-ODNs in each animal. In order to identify the sequences of CpG-ODNs that induce T(H)1 responses in dogs, mRNA expression and protein production of IFN-gamma were examined in peripheral blood mononuclear cells (PBMCs) from healthy dogs treated with 11 kinds of synthetic CpG-ODNs. One of the 11 CpG-ODNs (No. 2 CpG-ODN, 5'-GGTGCATCGATGCAGGGGGG-3') was shown to significantly increase mRNA expression and protein production of IFN-gamma in canine PBMCs in a manner dependent on the sequence of the CpG motif. This CpG-ODN also enhanced the expression of IL-12 p40 mRNA in canine PBMCs, whereas expression of IL-12 p35, IL-18, and IL-4 mRNAs was not induced by this CpG-ODN. These results indicate that this CpG-ODN was able to produce IFN-gamma by induction of T(H)1-skewed immune response in dogs. CpG-ODNs may be useful for inducing prophylactic and therapeutic immunity against allergic diseases, viral infection, and tumors in dogs.     

The incidence of disseminated intravascular coagulation in dogs with malignant tumor

The incidence of DIC in 208 dogs with a malignant tumor was evaluated. The incidence of DIC was 9.6% in dogs with a malignant tumor which was a solid tumor in all. In 164 dogs with a malignant solid tumor, the incidence of DIC was 12.2%. The incidence of DIC in dogs with hemangiosarcoma, mammary gland carcinoma and adenocarcinoma of the lung was significantly higher than that in dogs with other malignant tumors. These results suggested that special care in looking for DIC should be taken in dogs with a malignant solid tumor.     

Magnetic resonance imaging of alveolar echinococcosis experimentally induced in the rat lung

Pulmonary alveolar echinococcosis (AE) caused by the metacestode of Echinococcus multilocularis is a lethal zoonosis and is a lesion secondarily induced by hematogenous dissemination from hepatic AE lesions. In the present study, a hematogenous pulmonary AE model was experimentally induced in rats by the injection of echinococcal larval tissue homogenate to the tail vein, and then the pathological and diagnostic aspects of pulmonary AE were examined by magnetic resonance imaging (MRI). Histological primary, mature and degenerated AE lesions were observed 5, 18 and 50 weeks after injection, respectively. These lesions were discriminated as signal-void, hypointense and hyperintense regions in T1-weighted MRI (T1WI), respectively. The change in signal intensity in T1WI might reflect the content of proteinaceous fluid as a result of AE cyst degeneration. Western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis provided evidence for AE infection in the early stage. T1WI in combination with Western blot analysis could possibility become definitive and early signs of hematogenous pulmonary AE infection.     

Histopathological characteristics of Ito cells and Kupffer cells in the feline liver

The histopathological characteristics of Ito cells and Kupffer cells were investigated in the liver of 21 cats (age range: 6 months -18 years) autopsied in our laboratory during 2003. Immunohistochemical examinations were performed using antibodies against lysozyme, desmin and alpha-smooth muscle actin. No Kupffer cells reacted with the antibody against lysozyme. However, macrophages in the lung and spleen showed a positive reaction with the antibody. This finding suggests a possibility that the amount of lysozyme in the Kupffer cells of feline liver is comparatively small. On the other hand, large vacuole-laden cells were observed in the hepatic perisinusoid of some feline cases, and these cells showed a positive reaction with antibodies against desmin and alpha-smooth muscle actin. These cells could be Ito cells with large lipid vacuoles. This conclusion was supported by electron microscopic observation and oil red O staining. However, no such large vacuole-laden perisinusoidal cells were detected in the liver of young cats less than 2 years old. The present study revealed the histopathological features of Kupffer cells and Ito cells in the feline liver.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).     

Trapped neutrophil syndrome in a Border Collie dog: clinical, clinico-pathologic, and molecular findings

Trapped neutrophil syndrome (TNS) is an autosomal recessive inherited neutropenia known in Border Collies since the 1990's. Recently, the causative mutation has been identified in the canine VPS13B gene and a DNA-based diagnosis has now become available. The present paper describes clinical and clinico-pathologic findings in a Border Collie with TNS that was molecularly diagnosed for the first time in Japan. In a 10-week-old male Border Collie with microgenesis and symptoms related to recurrent infections, a hematological examination revealed severe leukopenia due to neutropenia, suggesting the dog to be affected by inherited neutropenic immunodeficiency. Direct DNA sequencing demonstrated that the dog was homozygous for the causative mutation of TNS and both its parents were heterozygous carriers. In addition, a simple and rapid polymerase chain reaction-based length polymorphism analysis coupled with microchip electrophoresis was developed for the genotyping of TNS. This assay could discriminate clearly all genotypes, suggesting that it was suitable for both individual diagnosis and large-scale surveys for prevention.     

Plasma Profiles of Glucose, Insulin and Lipids in the Male WBN/Kob-Lepr(fa) Rat, a New Model of Type 2 Diabetes with Obesity

Plasma profiles of glucose, insulin and lipids were examined in the male WBN/Kob-Lepr(fa) (fa/fa) rat, a new model of type 2 diabetes (T2D), in comparison with age-matched original male WBN/Kob (lean) rats. The fa/fa rats developed hypertriglycemia, obesity and hyperglycemia from 5, 7, and 9 weeks of age, respectively. Plasma insulin levels in fa/fa rats were significantly higher than those in lean rats at 5 weeks of age, but after 11 weeks of age gradually declined to the levels in lean rats. HOMA-IR, a measure of insulin resistance status, showed that fa/fa rats had insulin resistance. The fa/fa rat has the potential to become an important animal model of T2D with obesity.     

Effects of soil scraping on the reclamation of tsunami-damaged paddy soil

We investigated the possibility of tsunami-damaged paddy soil remediation in Japan using a scraping method. We collected undisturbed soil blocks to maintain the soil structure from paddy damaged by salt from the tsunami. Scraping treatments to remove the top 2 cm of the soil surface were conducted once, twice, and thrice after drying naturally in a greenhouse environment, as well as a noscraping control. Electrical conductivity (EC) of the soil dramatically decreased to < 1.36 dS m^?1 in the residual soils after scraping. The EC values of the residual soils were significantly lower after being scraped twice or thrice compared with a single scraping. Similarly, the mean NaCl removal rates were significantly higher with two (91%) or three scrapings (97.2%) compared with a single scraping (73.3%). The growth of rice plants was almost normal in the residual soils based on a visual score (approximately 3 on average) using a standard evaluation system at 4 weeks after transplantation. There were no significant differences in the grain yields from the residual soils with different scraping frequencies, and the EC values of all the residual soils were < 1.36 dS m^?1. This study clearly indicates that there were reductions in the EC and good plant growth performance in the residual soils after scraping, and there was also a high rate of NaCl removal. Therefore, scraping may be a useful method for the remediation of salt-damaged paddy soils when irrigation or culvert systems are affected severely by tsunamis and earthquakes.